Summary: More than 100 people in the UK and four in France have been diagnosed with a new TSE disease called variant Creutzfeldt-Jakob disease (vCJD) and possibly tens of thousands more may have been infected because of being exposed to bovine spongiform encephalopathy (BSE) contaminated meat. The TSE diseases have a long latent asymptomatic phase during which infected individuals may donate blood and potentially spread the disease. Because the infectivity of blood in rodents with experimental TSE is largely cell-associated, several countries have adopted a precautionary policy to universally leukoreduce (ULR) their blood products in order to decrease the theoretical risk of transmitting TSE disease by blood. Scientific data supporting this decision is not available, and the FDA has so far not recommended leukoreduction for this purpose. Information on whether ULR is effective in reducing TSE infectivity in blood and blood products is essential to support future regulatory decisions. Background: Very little actual data to support the efficacy of leukoreduction in clearance of TSE infectivity from blood is available. A single published leukofiltration experiment with fresh mouse plasma (Brown P et al. Transfusion, 1999) showed no effect on the level of TSE infectivity after filtration. Theoretically, filtration might even have the opposite of the intended effect, since leukocytes that fragment during the filtration process would release TSE infectivity into the plasma. We previously demonstrated that TSE infectivity in hamster blood is not associated with platelets, but is present in peripheral mononuclear cells. We plan to filter hamster blood to decrease the level of leukocytes and to follow endogenous TSE infectivity before and after filtration. TSE infectivity is propagated in the spleen of the hamster, so we plan to demonstrate the efficacy of leukoreduction on decreasing TSE infectivity by spiking separated splenocytes into whole blood and then filtering, titrating infectivity in the spiked starting material, the filtrate and the retentate. Methods: We plan to utilize the hamster TSE (scrapie) model and to study the effect of leukofiltration on endogenous infectivity present in platelet concentrates. Blood from a group of infected rodents will be pooled and then processed by the same methods used for human blood. Platelet concentrates will be prepared from pooled blood and leukofiltered using conventional platelet leukodepletion filters. Cell counts, membrane fragment counts and TSE infectivity levels will be assayed before and after leukofiltration. Leukoreduction filters for red cells usually have a large "dead space" and to fill that space would require an impossibly large pool of rodent blood. We plan to utilize hamster splenic leukocytes, collected from the same animals used for the platelet experiments, to "spike" human red blood cell units. TSE infectivity is propagated in the hamster spleen, which should be a rich source of infected leukocytes. The human red cells will then be filtered. Blood cells will be counted using a cell counter and the presence of membrane fragments will be evaluated using flow cytometry. TSE infectivity in fractions before and after leukofiltration will be evaluated by end point titration after intracerebral inoculation into hamsters. This is a standard bioassay method for detecting TSE infectivity. The presence of TSE disease in infected animals will be confirmed by histopathology of animal brains done in collaboration with NCTR. We plan to compare different filters marketed by several manufacturers. (Separated scrapie-infected cerebral cells, known to contain extremely large amounts of infectivity, will also be spiked into human blood and filtered as an additional check on the performance of the filters.) Approximately 1000 rodents will be used for the total experiment. Currently there is no practical method, other than bioassay, for measuring TSE infectivity in blood. The level of sensitivity in for an assay should be in the range of 10 pg PrPc/ml of whole blood. A number of assays have been developed that detect PrPc in brain but their sensitivity is not sufficient for use in blood. Additional concentration or amplification steps have recently been published and include PrPc precipitation with phosphotungstanate, selective binding of PrPsc with plasminogen and sonication-mediated amplification of PrPsc. In collaboration with the ORA, Denver district, we plan to modify these methods to develop an ELISA-based assay for detecting PrPsc in hamster blood and urine collected in the leukoreduction experiments and compare it to infectivity results obtained with bioassay. Feasibility and time frame: All methods required to conduct this project are already developed. The co-investigators have a track record of conducting TSE infectivity studies and studies of prion protein expression on blood cells. The study will take approximately half year to accomplish but the results of the infectivity bioassays, due to the long incubation period of the disease in animals will not be available for up to one year after inoculation. The collaboration with ORA will allow for testing of the novel diagnostic assays without sacrificing additional animals. Anticipated results: We anticipate that leukoreduction by filtration will significantly reduce TSE infectivity in blood products based on our previous observations that the major portion of infectivity found in hamster blood is associated with peripheral mononuclear leukocytes. We think it unlikely that leukoreduction will increase the risk of spreading TSE infectivity by releasing leukocyte fragments into plasma.